Biological systems consist of inherent chiral components, f.i. proteins, which may interact with the stereoisomers of endogenous and exogenous chiral compounds in different manners. As a result, stereoisomers of exogenous compounds such as drug substances, toxins, agricultural chemicals or food additives very often possess different pharmacological and toxicological profiles. This effect, for instance, forced developers of drug substances to study the pharmacological profiles of individual enantiomers separately and also resulted in an increased number of drug substances which are produced and marketed as an individual enantiomer. A precondition for that is the availability of preparative methods for the production of enantiomers and of analytical tools for the stereoselective analysis of quality and pharmacokinetic effects.
Individual enantiomers can be produced by stereoselective synthesis or racemate resolution. The latter approach is often the preferred and more widely available process, since syntheses of racemates can usually be carried out easily and at relatively low costs in comparison with a stereoselective synthesis and the methodologies of racemate resolution are often applicable to different, structurally varied classes of compounds. The latter approach can also be preferable if both enantiomers are used in purely enantiomeric form such as for pharmacological tests. In any case, the latter approach requires chiral selectors or functional materials that are modified by selector groups which can stereoselectively interact with both enantiomers if contacted with the racemate.
The present invention now discloses novel chiral selectors of that kind and novel functional materials, which may be applied in different preparative racemate resolution or enantiomeric separation concepts including chromatographic solid-liquid or liquid-liquid methods, solid-liquid or liquid-liquid extraction technologies and membrane separation techniques. Similarly, the chiral selectors and functional materials can also be used in analytical enantiomer separation methods, wherein they are integrated as adsorption materials in a column liquid chromatography, supercritical fluid chromatography, capillary electrochromatography, in chip technologies or as molecular recognition materials and sensitive layers in sensor technologies.
The enantioselective cation-exchange material according to the invention comprises a chiral selector composed of a chiral component and at least one cation-exchange group, a spacer and a carrier and is characterized in that the chiral component has a molecular weight of less than 1,000 and the at least one cation-exchange group is an acid group having a pKa<4.0.
The enantioselective cation-exchange material is preferably characterized in that the acid group has a pKa<3.5. Furthermore, it is preferred that the acid group has a pKa<2.5.
In a further preferred embodiment, the enantioselective cation-exchange material according to the invention is characterized in that the acid group is a sulfonic, sulfinic, phosphoric, phosphonic or phosphinic group.
The present invention is based on the surprising finding that the capabilities of chiral recognition of enantioselective cation exchangers improve with an increasing acidity of the low-molecular weight synthetic chiral selector.
The primary target compounds of the presently invented enantioselective molecular recognition materials of the cation-pairing type and the cation-exchange type are basic chiral drug substances and intermediates (chirons). Since a high percentage of the total market share of chiral drug substances contains basic functional groups, rendering them amenable to separation by the invented functional molecular recognition materials, those substances should have a wide range of applicability.
Moreover, many biomolecules are also chiral, comprising basic functionalities, and thus the presently invented functionalized solid phases and materials, respectively, are also suitable for separations of basic biomolecules, in particular of closely related analoga and isomeric forms. Application areas of the novel materials therefore also include the separation of amino acids, peptides, proteins, nucleotides, aminoglycosides and a large number of other basic chiral compounds.
In the following, the most important features of the present invention are described briefly. The present invention relates to enantioselective molecular recognition materials of the cation-exchange type and the cation-pairing type, composed of at least 3 modules which are i) an acidic chiral selector composed of a chiral component or a structural unit comprising a functional acid group, ii) a spacer and iii) a carrier or a polymer backbone, according to the general pattern as illustrated in FIG. 1.
In any case, the invented functional materials possess at least 1 free functional acid group (in the following referred to as X-substituent). The X-substituent is a strongly acidic functional group having a pKa<4.0 (based on an entirely aqueous system; regarding the conditions of pKa-determination, see Example 7) such as a sulfonic, sulfinic, phosphoric, phosphonic, phosphinic, boronic, amidophosphonic, amidosulfonic acid or any other acid group. Seen from the point of view of molecular recognition, this functional acid group controls the interaction with the cationic target, the analyte or the sample components via strong intermolecular ionic interactions in accordance with ion-pairing and ion-exchange mechanisms, which are the stronger, the lower the dielectric constant of the medium, but which are also active in aqueous or aqueous-organic liquid phases.
It has been found that the selector-analyte binding strength increases as the pKa of the cation-exchange group decreases, i.e. as the acidity of the functional acid group of the selector increases. Surprisingly, also the capability of enantioselective binding and separation improved with a stronger bond. This was by far not expected: Although it may be assumed that a stronger cation exchanger would bind oppositely charged analytes more strongly than a weak counterpart, higher enantioselectivity could not be expected as the logical consequence. On the contrary, experience shows that analytes which are bound too strongly very often produce only a moderate enantioselectivity.
The chiral component carrying the acidic X-substituent is a chiral compound in a single enantiomeric form or is constructed from enantiomerically pure chirons. Together, the chiral component and the X-substituent form the low-molecular weight chiral acidic selector forming the core of the present functional materials, as they provide the information for a selective molecular recognition of the target compounds. In contrast to macromolecular formations such as proteins, the chiral component preferably has a low molecular weight and is typically synthesized from chirons such as natural or unnatural, cyclic or non-cyclic amino acids, hydroxycarboxylic acids, aminophosphonic acids, aminophosphinic acids, aminosulfonic acids, aminosulfinic acids, aminoboronic acids, hydroxyphosphonic acids, mercapto-phosphonic acids, hydroxyphosphinic acids, tartaric acid derivatives, mandelic acid derivatives, camphor sulfonic acid derivatives, linear or cyclic, natural and unnatural peptides, linear or cyclic sulfopeptides, linear or cyclic phosphonopeptides. The low-molecular weight chiral acidic selector may also be an amphoteric compound which is negatively charged if used under conditions below the isoelectric point. Apart from the primary ionic interaction site created by the X-substituent, optimally functioning variants of molecular recognition materials of the cation-exchange type and the cation-pairing type, in addition, also exhibit other interaction sites such as hydrogen-donor-acceptor groups (indicated by Y) and/or π—π interaction sites (aromatic groups with preferably electron-withdrawing or electron-releasing substituents) as well as bulky elements for repulsive and/or attractive interactions of the van der Waals type. The functional Y-substituent may be an amide, carbamate, sulfonamide, urea, carbonyl, semicarbazide, hydrazide or sulfonimide group or another similar hydrogen-donor-acceptor system. Bulky elements of the selector component and interacting functional groups are assembled such that a binding pocket is preformed, with the polar interaction sites being arranged closer to the centre of the pocket and the hydrophobic bulky groups being arranged at the edge of the pocket. Target analytes which sterically and electrostatically meet the binding conditions are able to bind selectively into said pocket, whereas others are excluded from strong binding.
For many applications, those selectors must be immobilized on solid or optionally also liquid matrices referred to as carriers in such a way that the selector groups are properly exposed to the surrounding solution containing the target compounds to be selectively recognized and bound, whereby their binding is rendered possible. The carrier should be inactive (inert) with respect to the binding of the target compound, while having the function of guaranteeing the chemical and physical stability of the molecular recognition material. In flow applications such as chromatography, the carrier or its physical properties, respectively, determine(s) the kinetic properties of the materials. It is therefore an important component of the functional material. In the present invention, the carrier can be an inorganic, organic or mixed inorganic-organic hybrid-like material. Such carrier materials comprise commercially available and self-developed beads, monolithic or continuous materials, nanoparticles, membranes, resins, surface-limited layers produced from chemical materials comprising silica (SiO2), alumina (Al2O3), zirconia (ZrO2), titania (TiO2), materials derived from sol-gel, organic-inorganic silica-containing hybrid materials, optionally cross-linked polysiloxanes, any polymer obtained from vinyl monomers, optionally cross-linked poly(meth)acrylates, optionally cross-linked poly(meth)acrylamides, optionally cross-linked polystyrenes, mixed styrene-(meth)acrylate polymers, ring-opening methathesis polymers, polysaccharides, agarose and any of those materials specifically functionalized to permit immobilization of the low-molecular weight chiral acidic selector. Among preferred carriers, there are silica beads, poly(meth)acrylate polymer beads, poly(meth)acrylamide beads, poly(meth)acrylate monoliths, polystyrene resins which optionally are modified with adherent reactive groups in order to immobilize the selector.
A polymer backbone or a polymer matrix can also be regarded as a carrier so that a spacer and a chiral acidic selector represent actually adherent chiral groups of the polymer that are responsible for a selective molecular recognition of the basic target compounds.
The spacer has mainly the function of binding the low-molecular weight acidic selector to the carrier. Both the length and the chemical functionality of the spacer are variable. To a certain degree, it can participate in the selector-analyte binding, involving a positive, but also a negative effect on selectivity. It can influence the rigidity and accessibility of the selector, thereby also affecting the binding characteristics. Finally, it determines the chemical stability of the functional materials and can influence their compatibility with analytes, a factor which is essential in particular for the separation of biomolecules. All general solid-phase linker concepts and approaches for the preparation of chromatographic stationary phases can be used for the synthesis of the present functional materials. Immobilization strategies that are preferred for the synthesis of presently invented chiral cation-exchange materials comprise the reaction of a vinyl-modified acidic selector with a thiol-modified carrier, in particular with thiolpropyl-modified silica, via a radical addition reaction. Other immobilization concepts which may be applied comprise the asymmetrical reaction of a diisocyanate linker with an amino- or hydroxyalkyl-modified carrier and an amino- or hydroxy-modified selector component, the reaction of an amino-, hydroxy- or thiol-modified carrier with a chloro- or bromoalkanoyl-derivatized selector, the reaction of alkoxy- or chloroorganosilane with a terminal reactive functionality for coupling to a selector component, the hydrosilylation reaction of alkoxy- or chlorohydrosilane with a selector containing a vinyl group, the coupling of an amino-modified carrier and an amino-modified selector by reacting one of the 2 components with a dicarboxylic-acid anhydride spacer component and the subsequent activation of the resulting carboxylic acid function and reaction with a second amino component and many other immobilization strategies which in general are used for the immobilization of chiral selectors, proteins and peptides on solid carriers.
In special cases, the current enantioselective separation method of the cation-exchange type can be combined with other separation methods such as reversed-phase chromatography, for example, by using novel surface modifications obtained by combining the above-described selector components with long-chain alkyl groups, resulting in RP-chiral hybrid selectors of the cation-exchange type and solid phases with a dedicated selectivity and separation character.
One of the particular advantages is that the above-described functional materials are resistant to more or less all liquid-phase systems that are normally used. Hence, those functional materials can be operated with phenotypes of the aqueous, aqueous-organic (reversed phase), polar organic phase and apolar organic phase (normal phase), wherein the liquid or mobile phase must in any case contain a cation, preferably an ammonium or an organic ammonium compound, as a counterion, for operation in the ion-exchange mode.
The present invention also relates to the application of any method which uses the novel molecular recognition materials according to the invention of the cation-exchange type and the cation-pairing type in preparative chromatographic solid-liquid or liquid-liquid methods, solid-liquid or liquid-liquid extraction technologies and membrane separation techniques. In analogy, a further subject of the present patent application is their application in analytical methodologies, wherein they are integrated as adsorption materials in a column liquid chromatography, supercritical fluid chromatography, capillary electrochromatography, in chip technologies or as molecular recognition materials and sensitive layers in sensor technologies.